Minimization of a Protein−DNA Dimerizer

Stafford, Ryan L.; Arndt, Hans‐Dieter; Brezinski, Mary L.; Ansari, and Aseem Z.; Dervan, Peter B.

doi:10.1021/ja067971k

Cited by 22 publications

(15 citation statements)

References 68 publications

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“…A polyamide was conjugated to a dipeptide derived from the Hox protein known to interact with Exd. The Hox mimic was able to cooperatively interact with Exd and bind to specific DNA sequences with nanomolar affinity [105, 107]. …”

Section: Future Directionsmentioning

confidence: 99%

Small-molecule regulators that mimic transcription factors

Rodríguez‐Martínez

Peterson‐Kaufman

Ansari

2010

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Transcription factors (TFs) are responsible for decoding and expressing the information stored in the genome, which dictates cellular function. Creating artificial transcription factors (ATFs) that mimic endogenous TFs is a major goal at the interface of biology, chemistry, and molecular medicine. Such molecular tools will be essential for deciphering and manipulating transcriptional networks that lead to particular cellular states. In this minireview, the framework for the design of functional ATFs is presented and current challenges in the successful implementation of ATFs are discussed.

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Section: Future Directionsmentioning

confidence: 99%

Small-molecule regulators that mimic transcription factors

Rodríguez‐Martínez

Peterson‐Kaufman

Ansari

2010

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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“…Biophysical Journal 96(11) 4661-4671 a b-alanine residue and a dimethylaminopropylamide tail at the C-terminus each confer a specificity for A$T and T$A basepairs (31). This general addressability of the DNA minor groove is supported by x-ray and NMR structure studies (32,33) and has been utilized in several applications, including, for example, DNA nanostructures (34,35), recruitment of DNA-binding proteins (36,37), and the inhibition of gene expression within living cells (38)(39)(40).…”

Section: Introductionmentioning

confidence: 98%

Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

Dose

Albrecht

et al. 2009

Biophysical Journal

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Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA.ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA.ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA.ligand complexes.

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“…[18][19][20][21] In particular, with laser microscopy it is possible to visualize the uptake of polyamidefluorophore conjugates as they traffic unaided to the nucleus of live cells. [3][4][5][6] This advance has helped to usher in an era of gene regulation.…”

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Quantitating the concentration of Py-Im polyamide–fluorescein conjugates in live cells

Hsu

Dervan

2008

Bioorganic & Medicinal Chemistry Letters

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Quantitative fluorescence-based methods have been developed to determine the nuclear concentration of polyamide-fluorescein conjugates in cell culture. Confocal laser scanning microscopy and flow cytometry techniques are utilized to plot calibration curves, from which the nuclear concentration can be interpolated. Upon treatment with polyamide, the concentration in the nucleus of live HeLa cells is calculated to be between 0.1-0.5 μM, which is significantly lower than the 2 μM dosage concentration. In contrast, the observed nuclear concentration in U251 cells is closer to the dosage concentration, indicating a cell line-specific increase in uptake for this class of compounds. Although confocal microscopy and flow cytometry generate disparate values, taken together these experiments suggest that the polyamide concentration inside the cell nucleus is lower than it is outside the cell.Keywords gene regulation; fluorescence; confocal microscopy; flow cytometry Hairpin pyrrole-imidazole polyamides are synthetic ligands that target predetermined DNA sequences with affinities comparable to those of naturally occurring DNA-binding proteins. 1,2 These cell-permeable small molecules have been shown to localize to the nucleus of living cells 3-7 and regulate endogenous gene expression. 8-12 Polyamides selectively bind in the minor groove of DNA according to a set of "pairing rules," where each heterocyclic ring pair targets a specific Watson-Crick base pair. 1,2 The antiparallel pairing of N-methylpyrrole (Py) and N-methylimidazole (Im) aromatic rings targets the C·G base pair, while Im/Py discriminates G·C. 13-16 The Py/Py pair recognizes A·T and T·A, while chlorothiophene can be paired with Py to distinguish T·A at the N-terminus. 17Polyamide-small molecule conjugates have been utilized in a number of applications. 18-21 In particular, with laser microscopy it is possible to visualize the uptake of polyamidefluorophore conjugates as they traffic unaided to the nucleus of live cells. 3-6 This advance has helped to usher in an era of gene regulation. Figure S1), confocal laser scanning microscopy images (Supplementary Figure S2), and experimental details (Supplementary Information).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. scoring system to rate the extent of nuclear localization in cell culture. 5,6 Upon incubation with polyamide and direct imaging of cells, the fluorescence intensity in the nucleus is compared to that in the medium. Positive scores are assigned when the nuclear staining exceeds that of the medium. The degree of nuclear localization varies across p...

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Minimization of a Protein−DNA Dimerizer

Cited by 22 publications

References 68 publications

Small-molecule regulators that mimic transcription factors

Small-molecule regulators that mimic transcription factors

Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

Quantitating the concentration of Py-Im polyamide–fluorescein conjugates in live cells

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